In recent years, the development of alternative energy to oil is a very important issue, because of environmental problems, such as global warming and aerial pollution, in addition to the concern related to transportation energy supply. Plant biomass is the most abundant renewable energy source on earth, which is expected to serve as an alternative source to petroleum. Lignocellulose is the main component of plant biomass and is composed of polysaccharides such as celluloses and hemicelluloses (including xylan, arabinan and mannan), lignin and other pectins. These polysaccharides are hydrolyzed into monosaccharides such as glucose and xylose by a variety of glycoside hydrolases, and are used as a biofuel or a raw material of chemical products.
Lignocellulose having a complex structure is persistent, and is difficult to degrade or hydrolyze with a single enzyme. For this reason, the hydrolysis of cellulose among the polysaccharides generally requires three types of enzymes: an endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21) that are glycoside hydrolases. On the other hand, hemicellulose contains xylan, arabinan, mannan and the like, and the composition thereof depends on the type of the plants. For example, xylan is a major constituent in broad-leaved trees, herbaceous plants and the like. For the hydrolysis of xylan, it is thought that xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) are required.
In the conventional lignocellulose to ethanol conversion process, high-solid loading up to 30-60% in initial substrate concentration has been attempted for the purpose of higher energy efficiency and less water usage. The enzymatic hydrolysis of lignocellulose by such high-solid loading processes results in the high viscosity of the hydrolyzed biomass solution so that the hydrolysis of lignocellulose hardly proceeds. Therefore, for example, by carrying out the enzymatic hydrolysis process at a high temperature of 80° C. or higher using a thermostable enzyme, in addition to an increase in the hydrolysis reaction rate, since the viscosity of the hydrolyzed biomass solution also reduces, the shortening of the hydrolysis reaction time and the reduction of the amount of enzyme are expected to be achieved. For this reason, for various glycoside hydrolases, development of enzymes that are more excellent in terms of thermostability has been desired.
Many thermostable glycoside hydrolases have been obtained by isolating and identifying the thermophilic microorganisms that live in a high temperature environment, cloning the genes from these cultured and isolated microorganisms and determining the DNA sequence thereof, followed by the expression thereof using Escherichia coli, filamentous fungi and the like. For the purpose of lignocellulose degradation, use as a processing agent of cellulose fibers, and pulp and paper processing, numerous thermostable enzymes that can be used for lignocellulose hydrolysis treatment at a high temperature, especially endoglucanases necessary for the hydrolysis of cellulose, have been isolated to date from thermophilic bacteria, filamentous fungi, archaea and the like (for example, see Patent Documents 1 to 4, and Non-Patent Documents 1 and 2). In addition, attempts have also been made to further improve the specific activity and the heat resistance, for example, by using the mutants of a host organism or partially modifying the amino acid sequences of these enzymes (for example, see Non-Patent Documents 3 and 4). However, most of these enzymes have an optimum temperature of 60 to 80° C., and a further increase in the degree of heat resistance has been desired. On the other hand, hyperthermostable endoglucanases having an optimum temperature of more than 90° C. have been reported (for example, see Patent Documents 5 to 7, and Non-Patent Documents 1 and 5). According to Non-Patent Document 5, an endoglucanase of Sulfolobus solfataricus having an optimum temperature of 95° C., has been reported, although the specific activity thereof is 5.5 U/mg protein at 90° C., which is not high. The above enzyme also has xylanase activity, although the specific activity of xylanase is 4.0 U/mg protein 90° C., which is also low.